The myc proto-oncogenes encode transcriptional regulators whose inappropriate expression is correlated with a wide array of malignancies. At the cellular level Myc activity has been linked with cell division, accumulation of mass, differentiation, and programmed cell death. The molecular mechanisms by which these end points are achieved have been show to emerge. It is now clear that Myc can directly influence the expression of hundreds, perhaps thousands, of genes with diverse functions. A significant challenge for the future will be to integrate this wealth of information into mechanistic models that explain the biological functions of Myc. This proposal is centered on a genetic analysis of cellular functions controlled by Myc. Three c-myc loss-of-function model systems will be used: an already existing c-myc knockout in the Rat-1 fibroblast cell line, mouse embryo fibroblasts (MEF) derived from conditionally targeted c-myc mice, and human c-myc knockout fibroblasts, whose construction and analysis constitutes aim 1 of this proposal. In all three models conditional Myc expression will be engineered on the knockout background. Aim 2 will analyze mutations in c-Myc effector domains that will be knocked into one copy of the endogenous c-myc gene. After turning off the wild-type c-myc gene copy, cells will be examined for known Myc phenotypes. Expression of individual target genes will be studied in detail, biochemical functions known to be affected by Myc will be assayed, and interaction of the mutant Myc proteins with known partners will be determined. Aim 3 will reconstitute functional expression of selected Myc target genes in the knockout cell lines. Myc-activated genes will be overexpressed using retrovirus vectors, and Myc-repressed genes will be ablated using RNAi. Phenotypes of these interventions will be studied, and multiple interventions will be attempted in combination. Aim 4 will determine the mechanisms by which Myc promotes cell cycle entry. D-type cyclin expression and activation of Cdk4/6 will be examined in c-myc/- MEF, rescue with a constitutively active Cdk4 will be attempted, the presence or absence of rescue will be analyzed in molecular terms, and posttranslational mechanisms leading to thr activation of cyclin D-Cdk4/6 complexes will be examined. These studies are expected to shed light on how specific outputs, such as the promotion of cell growth or cell Cycle progression, are causally associated with specific cellular targets and molecular mechanisms. [unreadable] [unreadable]